Development and launch of the first obstetrics and gynaecology master of medicine residency training programme in Botswana.

BACKGROUND: Sub-Saharan Africa (SSA) faces a severe shortage of Obstetrician Gynaecologists (OBGYNs). While the Lancet Commission for Global Surgery recommends 20 OBGYNs per 100,000 population, Botswana has only 40 OBGYNs for a population of 2.3 million. We describe the development of the first OBGYN Master of Medicine (MMed) training programme in Botswana to address this human resource shortage. METHODS: We developed a 4-year OBGYN MMed programme at the University of Botswana (UB) using the Kern's approach. In-line with UB MMed standards, the programme includes clinical apprenticeship training complemented by didactic and research requirements. We benchmarked curriculum content, learning outcomes, competencies, assessment strategies and research requirements with regional and international programmes. We engaged relevant local stakeholders and developed international collaborations to support in-country subspecialty training. RESULTS: The OBGYN MMed curriculum was completed and approved by all relevant UB bodies within ten months during which time additional staff were recruited and programme financing was assured. The programme was advertised immediately; 26 candidates applied for four positions, and all selected candidates accepted. The programme was launched in January 2020 with government salary support of all residents. The clinical rotations and curricular development have been rolled out successfully. The first round of continuous assessment of residents was performed and internal programme evaluation was conducted. The national accreditation process was initiated. CONCLUSION: Training OBGYNs in-country has many benefits to health systems in SSA. Curricula can be adjusted to local resource context yet achieve international standards through thoughtful design and purposeful collaborations.

Clinical use of blood and blood components in post-abortion care in Botswana.

BACKGROUND: Understanding the pattern and gaps in blood product utilisation in post-abortion care is crucial for evidence-based planning and priority setting. OBJECTIVE: To describe the current use of blood and blood components in post-abortion care in Botswana. METHODS: We conducted a retrospective cross-sectional study across four hospitals in Botswana using routine patients' records. We included all patients admitted for an abortion between January and August 2014. Descriptive statistics are used to report the results. RESULTS: Whole blood and red cell concentrates were used in 59/619 (9·5%) of patients. Plasma and platelet use was 1·3 and 0·7%, respectively. The mean admission haemoglobin level was 10·07 g dL(-1) (SD 2·69) and differed significantly between referral and district hospitals. The mean number of blood units transfused per patient was 2·23 (standard deviation (SD) 1·23), with 15/55 (27·3%) receiving a single unit. A total of 43/288 (14·9%) of the patients had haemoglobin levels below 7 g dL(-1) but did not receive any transfusion. There was a moderate positive correlation between admission haemoglobin level and time to transfusion (Spearman's rho = 0·37, P = 0·01). The number of blood units given increased with decreasing admission haemoglobin level. The strength of this association was moderate (Spearman's rho = -0·48, P < 0·001). CONCLUSION: There is a relatively low utilisation of blood and blood components in post-abortion care in Botswana despite an apparent clinical need in some instances. The reason for this shortfall, as well as its impact on morbidity and mortality, needs to be explored and be a focus of health systems research in Botswana.

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast.

Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored. Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts. Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP. The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export.

The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A)+ RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.

A role for the divergent actin gene, ACT2, in nuclear pore structure and function.

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.

Yeast mutants that produce a novel type of ascus containing asci instead of spores.

Tetraploid yeast cells lacking BFR1 or overexpressing an essential gene BBP1 produce a novel type of ascus that contains asci instead of spores. We show here that the asci within an ascus likely arise because a/alpha spores undergo a second round of meiosis. Cells depleted of Bbp1p or lacking Bfr1p are defective in a number of processes such as nuclear segregation, bud formation, cytokinesis and nuclear spindle formation. Furthermore, deletion of BFR1 or overexpression of BBP1 leads to an increase in cell ploidy, indicating that Bfr1p and Bbp1p play roles in both the mitotic cell cycle and meiosis. Bfr1p and Bbp1p interact with each other in a two hybrid assay, further suggesting that they might form a complex important for cell cycle coordination.

gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.

The nucleolus: an organelle formed by the act of building a ribosome.

Recent evidence corroborates the idea that the structure of the nucleolus need not be strictly maintained for proper function, suggesting that the organelle is composed of supramolecular assemblies formed during rRNA synthesis. More controversial is whether the nucleolus exists in the absence of rRNA synthesis and whether it interacts with the nuclear scaffold. The simultaneous and highly integrative nature of building a ribosome is reflected in the numerous observations showing that proteins involved in all aspects of ribosomal biogenesis affect pre-rRNA processing. The identification of several new nucleolar proteins without an obvious role in pre-rRNA metabolism may provide the field with long sought after assembly factors that might be key players in eukaryotic ribosome biogenesis.

Nucleolar proteins that bind NLSs: a role in nuclear import or ribosome biogenesis?

In a search for proteins that bind nuclear localization sequences (NLSs), a number of nucleolar proteins with diverse functions were found. It is thought that the assay fortuitously uncovered a novel domain that mediates the interaction between these nucleolar proteins and ribosomal proteins containing NLS-like sequences. The domain is highly acidic and contains a number of serines forming putative casein kinase II sites. Here, we propose a model in which the nucleolar proteins catalyse the assembly of ribosomal proteins with pre-rRNA.

Yeast NPI46 encodes a novel prolyl cis-trans isomerase that is located in the nucleolus.

We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Nopp140, and a COOH terminus with 45% homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cis-trans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

Multiple regions of NSR1 are sufficient for accumulation of a fusion protein within the nucleolus.

NSR1, a 67-kD nucleolar protein, was originally identified in our laboratory as a nuclear localization signal binding protein, and has subsequently been found to be involved in ribosome biogenesis. NSR1 has three regions: an acidic/serine-rich NH2 terminus, two RNA recognition motifs, and a glycine/arginine-rich COOH terminus. In this study we show that NSR1 itself has a bipartite nuclear localization sequence. Deletion of either basic amino acid stretch results in the mislocation of NSR1 to the cytoplasm. We further demonstrate that either of two regions, the NH2 terminus or both RNA recognition motifs, are sufficient to localize a bacterial protein, beta-galactosidase, to the nucleolus. Intensive deletion analysis has further defined a specific acidic/serine-rich region within the NH2 terminus as necessary for nucleolar accumulation rather than nucleolar targeting. In addition, deletion of either RNA recognition motif or point mutations in one of the RNP consensus octamers results in the mislocalization of a fusion protein within the nucleus. Although the glycine/arginine-rich region in the COOH terminus is not sufficient to bring beta-galactosidase to the nucleolus, our studies show that this domain is necessary for nucleolar accumulation when an RNP consensus octamer in one of the RNA recognition motifs is mutated. Our findings are consistent with the notion that nucleolar localization is a result of the binding interactions of various domains of NSR1 within the nucleolus rather than the presence of a specific nucleolar targeting signal.

The amino terminus of mammalian nucleolin specifically recognizes SV40 T-antigen type nuclear localization sequences.

Nucleolin is a major nucleolar protein in mammalian cells that is thought to be involved in ribosome biogenesis. The discovery that nucleolin shuttles between the cytoplasm and the nucleus raises the possibility that it is also involved in transporting ribosomal or nuclear proteins to the nucleus. The three structural domains of nucleolin bear a striking resemblance to the domains of a previously identified yeast protein NSR1, although the two proteins do not share a high degree of sequence similarity. NSR1 specifically recognizes the nuclear localization sequence (NLS) of both the simian virus large T antigen (SV40 T-antigen) and the yeast histone H2B by ligand blot analysis, and is a candidate for a receptor involved in the initial stages of nuclear transport. We report here that nucleolin, either purified from Chinese hamster ovary (CHO) cells or expressed in yeast, also specifically recognizes the wild-type, but not a mutant, histone H2B nuclear localization sequence by ligand blot analysis. The NLS recognition site is located within the N-terminal domain of both proteins. In showing that nucleolin, a protein that moves between the cytoplasm and the nucleus, also has the ability to interact with nuclear localization signals, our data support the idea that shuttling nucleolar proteins play a role in nuclear transport.

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs.

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.

Identification and characterization of a nuclear localization sequence-binding protein in yeast.

Nuclear proteins contain specific regions that are required for entry into the nucleus. Using ligand blotting, we have shown that a 67-kDa yeast nuclear envelope protein (p67) recognizes synthetic peptides containing the yeast histone H2B or simian virus 40 large tumor antigen nuclear localization sequence. Both free peptide and peptide conjugated to human serum albumin are recognized. The interaction between p67 and the nuclear localization sequences is specific; neither a mutant peptide that is incompetent for nuclear transport in vivo nor HSA can interact with p67 on blots. Moreover, although the wild-type peptide competes for binding to p67, the mutant peptides do not. p67 appears to be located at the nuclear envelope and is not present in other subcellular fractions. The nuclear localization sequence-binding protein is not extracted from the nuclear envelope with nonionic detergents and only partially extracted with high-salt buffer or 8 M urea, suggestive of a tight association with the nuclear envelope. Together our results are consistent with a role for p67 in nuclear transport.

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways.

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase.

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides.

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.